1. Field of the Invention
The present invention relates to methods for detecting the multidrug resistance phenotype in vivo and in vitro. The invention particularly relates to methods of diagnosing the multidrug resistance phenotype by imaging, particularly scintigraphic imaging, in solid tumors in vivo or in tumors and biopsies in vitro. The methods of the present invention allow the diagnosis of multidrug-resistant tumors and other multidrug-resistant phenotypes without invasive surgical methods.
The invention also relates to methods to increase the net uptake of drugs, and especially chemotherapeutic drugs, into cells and particularly to cells in vivo in malignant tumors. The invention is directed to new chemosensitizing agents and their use to enhance the uptake of various chemotherapeutic drugs. The methods of the present invention allow therapy of patients with multidrug resistant tumors. The methods of therapy of the present invention use compounds that interact with the multidrug resistance transport protein. The invention is directed to multidrug-resistance reversal agents that inhibit the cellular efflux of chemotherapeutic and other cytotoxic drugs in vivo and in vitro.
2. Description of the Background Art
A. ATP Binding Cassette Transport Proteins
(Higgins, C. F. et al., Bioessays 8:11 (1988); Ames, G. F., Ann. Rev. Blochem. 55:397 (1986); Higgins, C. F. et al., J. Bioenerg. Biomembr. 22:571 (1990); Hyde, S. C. et al., Nature 396:362 (1990); Higgins, C. F. et al., Phil. Trans. R. Soc. Lond. B 326:353 (1990)).
A large number of cellular proteins bind ATP, many of which utilize the free energy of ATP hydrolysis to drive particular biological reactions. Several of these comprise a subfamily, the members of which share considerable sequence homology. The region of homology extends over 200 amino acids. In several of the proteins the conserved domain comprises nearly the entire polypeptide. In others the conserved domain is only one segment of a multi-domain protein. These proteins include the multidrug resistance P-glycoprotein, the product of the White locus of Drosophila, procaryotic proteins associated with membrane transport, cell division, nodulation and DNA repair, the STE-6 gene product that mediates export of yeast .alpha.-factor mating pheromone, pfMDR that is implicated in chloroquine resistance of the malarial parasite, and the product of the cystic fibrosis gene (CFTR).
There are two short amino acid sequence motifs which are present in most if not all nucleotide binding proteins. The subfamily of ATP binding proteins that are relevant to this invention are distinct from all other ATP binding proteins in that they share considerably more sequence identity than is simply required for nucleotide binding. Other ATP binding proteins may possess the consensus nucleotide binding motifs but otherwise share no significant sequence similarity. This implies that the subfamily of proteins shares common functions in addition to the ability to bind ATP. Many of the proteins of the subfamily and those which are best characterized, are components of an active transport system which mediates the transport of molecules across the cytoplasmic membrane. They are recognized in the art as ATP-binding cassette superfamily of transport proteins (Hyde, S., et al., Nature 346:362 (1990)).
Several nucleotide binding protein-dependent transfer systems have been characterized in procaryotes. Each system requires a substrate binding protein located in the periplasm that provides a primary receptor for transport. The system also contains two integral membrane proteins that transport substrates across the membrane. The system further contains two peripheral membrane proteins thought to be located on the inner surface of the cytoplasmic membrane. These peripheral membrane proteins are members of the subfamily of bacterial and eucaryotic ATP binding proteins relevant to this invention.
P-glycoprotein is a eucaryotic four-domain protein consisting of two hydrophobic domains and two ATP binding domains. Besides the conserved ATP binding domains, there is a great deal of similarity between P-glycoprotein and bacterial binding protein dependent transport systems. The organization of this protein is remarkably similar to that of bacterial transport systems. The two hydrophobic domains in P-glycoprotein are homologous to each other. The same is true for the two hydrophobic components of the binding protein system.
There are also a number of differences. First, the P-glycoprotein consists of four domains encoded as a single polypeptide, whereas in bacteria the equivalent domains are on separate polypeptides. Second, P-glycoprotein pumps drugs out of the cell whereas the binding protein dependent transport system mediates uptake. Third, protein dependent transport systems require a periplasmic component which serves as the initial substrate binding site and delivers substrate to the membrane component. However, as far as is known there is no equivalent component which interacts with the P-glycoprotein. Fourth, there is an important difference between P-glycoprotein and the bacterial transport systems in substrate specificity. The bacterial systems are relatively specific and there is a separate system for each substrate. In contrast, P-glycoprotein exhibits a very broad specificity, handling a range of apparently unrelated drugs. However, most of the differences may be trivial rather than fundamental mechanistic differences. It is not uncommon for functions carried out by separate polypeptide chains in procaryotes to be fused into a single multifunctional protein in eukaryotes. Further, a small change in the organization of a transport system could promote efflux rather than uptake. Finally, the periplasmic components of bacterial systems can be viewed as a specific adaptation to the fact that bacteria have a periplasm.
The similarity between P-glycoprotein and the bacterial active transport system may be relevant to the mechanisms of multidrug resistance in eucaryotic cells. All available evidence is compatible with the view that P-glycoprotein is a eucaryotic transport system. Most chemotherapeutic drugs are lipophilic and can enter the cells passively. In multidrug-resistant cells, the intracellular concentration of these drugs is reduced in an energy-dependent manner. The most reasonable explanation for these findings is that P-glycoprotein is an active transport system, pumping drugs out of the cell.
B. Multidrug Resistance
One problem facing the cell biologist and oncologist is the tendency of cultured cells and tumors in patients to exhibit simultaneous resistance to multiple chemically unrelated chemotherapeutic agents. Tissue culture cells can be selected for resistance to a variety of drugs such as colchicine, doxorubicin (Adriamycin), actinomycin D and vinblastine. Increasing the concentration of the selecting agent in multiple small single steps results in high levels of cross resistance to these agents as well as many other drugs including other anthracyclines, Vinca alkaloids and epipodophyllotoxins (Gottesman, M. M. et al., J. Biol. Chem. 263:12163 (1988)).
Resistance of malignant tumors to multiple chemotherapeutic agents is a major cause of treatment failure (Wittes et al., Cancer Treat. Rep. 70:105 (1986); Bradley, G. et al., Biochim. Biophys. Acta 948:87 (1988); Griswald, D. P. et al., Cancer Treat. Rep. 65(S2):51 (1981); Osteen, R. T. (ed.), Cancer Manual, (1990)). Tumors initially sensitive to cytotoxic agents often recur or become refractory to multiple chemotherapeutic drugs (Riordan et al., Pharmacol. Ther. 28:51 (1985); Gottesman et al., Trends Pharmacol. Sci. 9:54 (1988); Moscow et al., J. Natl. Cancer Inst. 80:14 (1988); Croop, J. M. et al., J. Clin. Invest. 81:1303 (1988)). Cells or tissues obtained from tumors and grown in the presence of a selecting cytotoxic drug can result in cross-resistance to other drugs in that class as well as other classes of drugs including anthracyclines, Vinca alkaloids, and epipodophyllotoxins (Riordan et al., Pharmacol. Ther. 28:51 (1985); Gottesman et al., J. Biol. Chem. 263:12163 (1988)). Thus, acquired resistance to a single drug results in simultaneous resistance to a diverse group of drugs that are structurally and functionally unrelated.
The characteristics of the multidrug resistance phenotype have been analyzed by studies on normal and tumor cell lines isolated for resistance to selected cytotoxic drugs. One major mechanism of multidrug resistance in mammalian cells involves the increased expression of the 170-kDa plasma membrane glycoprotein, P-glycoprotein (for review, Juranka et al., FASEB J 3:2583 (1989); Bradley, G. et al., Blochem. Biophys. Acta 948:87 (1988)). Transfection of cloned P-glycoprotein genes into drug-sensitive cell lines has confirmed that an increased expression of P-glycoprotein is sufficient to cause multidrug resistance in experimental systems (i.e., Gros, P. et al., Nature 323:728 (1986)).
The nucleotide sequence of multidrug resistance cDNA (Gros, P. et al., Cell 47:371 (1986); Chen, C. et al., Cell 47:381 (1986)) indicates that it encodes a polypeptide similar or identical to P-glycoprotein and that these are members of the highly conserved class of membrane proteins similar to bacterial transporters and involved in normal physiological transport processes.
The multidrug resistance P-glycoprotein may function normally to extrude as yet unknown physiological substrates out of cells by an energy-dependent process (Arceci, R. J. et al., PNAS USA 85:4350 (1988)) in normal tissues. The gene is amplified and consequently overexpressed in malignant tissues. It is thus believed that by transporting chemotherapeutic agents out of the cells, P-glycoprotein renders tumors resistant to chemotherapy.
C. Visual Assay of Multidrug Resistance
Multidrug resistance has been detected in vitro in single cell suspensions and in cell monolayers. Yoshimura et al., (Cancer Letters 50:45 (1990)) used the uptake of rhodamine dye to screen for agents that overcome multidrug resistance in a cell line ("reversing agents"). The dye is accumulated in multidrug-resistant cells at a lower rate than it is accumulated in non-resistant cells and thus multidrug-resistant cells can be distinguished from non-resistant cells by comparing intracellular dye levels.
In this study, the authors monitored dye levels in multidrug-resistant cells in the presence and absence of verapamil, a known chemosensitizer (reversing agent used in chemotherapy to facilitate the uptake of a chemotherapeutic drug in drug-resistant tumor cells), and found that the dye accumulated to normal levels when the multidrug resistance phenotype was reversed with verapamil. The dye was administered to cells in a confluent monolayer. The cells were then either washed, solubilized, and the dye detected with a fluorescence spectrometer, or scanned in microtitre wells with a fluorescence microplate reader.
Efferth et al. (Arzneim-Forsch 38:1771 (1988)) also developed an in vitro assay to detect the multidrug resistance phenotype. They compared the levels of rhodamine dye in a cell sample with the levels of dye found in a control sample of normal cells. The dye was detected by forming a single cell suspension, pipetting the suspension onto slides, administering the dye to the cells on the slide and detecting dye uptake of cells on the slide.
Herweijer et al. (Invest New Drugs 7:442 (1989)) used on-line flow cytometry to detect cells with the multidrug resistance phenotype in a single cell suspension. The uptake kinetics of a fluorescent drug were measured on line first in the absence and then in the presence of a reversing agent.
Konen et al. (J. Histochem. Cytochem. 37:1141 (1989)) assayed efflux activity of the multidrug resistance transport system using fluorescence microscopy to monitor the accumulation of drugs in single cultured cells that were transformed with multidrug resistance DNA. They showed that the efflux pathway was inhibited when the cells were incubated with verapamil.
D. Scintigraphic Imaging with Hexakis (R-isonitrile) Technetium Complexes
Hexakis (R-isonitrile) technetium (I) complexes (where R is alkyl, substituted alkyl, aryl, or substituted aryl) are a class of low valence technetium (.sup.99m Tc) coordination compounds empirically designed as clinical myocardial perfusion imaging agents (Jones, A. G. et al., Int. J. Nucl. Med. Biol. 11:225 (1984), Holman, B. L., et al., J. Nucl. Med. 25:1350 (1984), Holman, B. L., et al., ibid 28:13 (1987), Sporn, V., Clin. Nucl. Med. 13:77 (1988)). Conceived to be used in a manner similar to thallus chloride for the noninvasive evaluation of coronary artery disease, the compounds exploit the more favorable emission characteristics of .sup.99m Tc for applications in clinical imaging (Strauss, H. W., et al., Radiology 160:577 (1986), Deutsch, E., et al., Science 214:85 (1981)). Chemical analysis of these complexes with the ground state .sup.99 Tc isotope shows them to be monovalent cations with a central Tc(I) core octahedrally surrounded by six identical ligands coordinated through the isonitrile carbon. The terminal R groups, when bound to the technetium, encase the metal with a sphere of lipophilicity (Jones, A. G., et al., Int. J. Nuc. Med. Biol. 11:225 (1984), Mousa, S. A., et al., J. Nuc. Med. 28:1351 (1987)).
These complexes are sufficiently lipophilic to partition into and through the hydrophobic core of biological membranes, but also combine this property with a delocalized cationic charge which renders the compounds responsive to the plasma and mitochondrial transmembrane potentials. This combination of lipophilicity and delocalized charge produces an unusual property for these pharmaceuticals. Unlike tissue binding of many other pharmaceuticals that depend on highly specific binding sites (high affinity receptors), these pharmaceuticals have a non-specific uptake mechanism. However, tissue interaction is highly specific for those tissues with high plasma membrane potentials, high mitochondrial membrane potentials, high mitochondrial content, or combinations of the above.
Because uptake of these compounds by tissues is non-specific, any living cell (and potentially, any tissue type) can retain the compounds. A further advantage is that the compounds have been shown to be safe in humans as diagnostic pharmaceuticals while maintaining the unique combination of properties that allow them to respond to membrane potential. Conversely, other classes of lipophilic cations or fluorescent probes of membrane potential (e.g., rhodamine 123) have been shown to be toxic to cells and mitochondria (Bernel, et al., Science 218:1117 (1982), Emaus, R. K., et al., Biochim. Biophys. Acta 850:436 (1986), Gear, A. R. L., J. Biol. Chem. 249:3628 (1974)). These compounds have not been injected into humans.
E. Model for P-Glycoprotein Function
Based on the information obtained from amino acid sequence analysis of P-glycoprotein from various mammalian cells, a model for P-glycoprotein function has been suggested (Bradley et al., Biochimica et Biophysica Acta 948:87 (1988)). The model suggests that P-glycoprotein forms a channel in the plasma membrane and transports drugs out of cells using energy derived from ATP hydrolysis. In one version of the model, P-glycoprotein binds drugs directly and then removes them from the cell. It is suggested that, since transfection of a P-glycoprotein cDNA clone into drug sensitive cells results in cross-resistance to structurally unrelated drugs, the P-glycoprotein molecule may have binding sites for a diverse group of drugs.
There is other experimental evidence to support a drug binding function for P-glycoprotein. First, membrane vesicles from drug resistant cell lines have been shown to overexpress a protein of 150-180 kilodaltons that specifically binds vinblastine (Safa et al., J. Biol. Chem. 261:6137 (1986); Cornwell et al., Proc. Natl. Acad. Sci. USA 83:3847 (1986)). Labeling is inhibited by drugs which are cross-resistant with vinblastine in these cells. This suggests that the drugs may be competing for either the same binding site or a closely adjacent binding site. The 150-180 kilodalton protein that binds vinblastine was immunoprecipitated with a monoclonal antibody against P-glycoprotein (Cornwell, M. M., et al., J. Biol. Chem. 262:2166 (1987)).
Further evidence that drug binding is involved in the function of P-glycoprotein in multidrug resistant cells is derived from the study of chemosensitizers/reversing agents (see also text below). Many of these agents inhibit photoaffinity labeling of P-glycoprotein by vinblastine analogs (Akiyama, S. I., et al., Mol. Pharm. 33:144 (1988)). It thus appears that the mechanism of action of reversing agents may be to inhibit toxic drug binding to P-glycoprotein.
However, it appears that there may be multiple drug binding domains in the P-glycoprotein because, first, the inhibition of the vinblastine analog binding was not equivalent to the ability to reverse multidrug resistance for several compounds, and, second, drugs that are involved in the multidrug resistance phenotype do not necessarily compete for the vinblastine analog binding site in vesicles from multidrug-resistant cells (Cornwell et al., M. M., et al., J. Biol. Chem. 262:2166 (1987); Akiyama, S. I., et al., Mol. Pharm. 33:144 (1988)).
In an alternative version of the model for P-glycoprotein function, a drug binding protein is transported out of cells by a P-glycoprotein pump. Drugs may bind irreversibly to this protein, and the entire drug-protein complex may then be removed from the cell.
Direct binding of drug analogs to the P-glycoprotein has been observed. P-glycoprotein can be labeled directly by a photoactive vinblastine analog in a saturable manner. This photoaffinity labeling can be inhibited by drugs such as daunomycin or vincristine, as well as several chemosensitizing agents such as verapamil, quinidine, reserpine, and azidopine (Gottesman, M. M., et al., Trends Pharmacol. Sci. 9:54 (1988)). Conversely, the labeling of P-glycoprotein by a photoactive analog of verapamil can be inhibited by some, but not all, drugs involved in the MDR phenotype (Safa, A. R., Proc. Natl. Acad. Sci. USA 85:7187 (1988)). Because these drugs and reversing agents may inhibit binding to P-glycoprotein, this suggests that a common binding site may be involved. Accordingly, a mechanism of reversal of the MDR phenotype by reversing agents/chemosensitizers may be explained on the basis of competition for drug binding, which results in decreased efflux of drugs which are taken up by the cell and thus a higher intracellular level of such drugs, such as chemotherapeutic drugs, in cells that are multidrug resistant.
F. Pharmacological Reversal of Multidrug Resistance by Chemosensitizers
Since recent studies have shown that P-glycoprotein associated multidrug resistance occurs clinically (Yoshimura, A., et al., Cancer Lett. 50:45 (1990)), strategies designed either to block expression or to circumvent this form of drug resistance are being sought by researchers in the field of cancer therapeutics. Particularly desirable are agents that inhibit P-glycoprotein activities at concentrations with little or no cytotoxic effect. These would be used in overcoming multidrug resistance when they are administered in combination with other anti-cancer drugs during clinical chemotherapy.
The following parameters have been used to screen agents that overcome multidrug resistance: (1) enhanced cytotoxicity of anti-cancer drugs to multidrug resistant cells; (2) enhanced accumulation of anti-cancer drugs in multidrug resistant cells; (3) inhibition of photoaffinity labeling of P-glycoprotein with photoanalogs of anti-cancer drugs and resistance modifiers; and (4) inhibition of binding of vinblastine or vincristine to membrane vesicles from multidrug resistant cells (Yoshimura et al., A., et al., Cancer Lett. 50:45 (1990)).
The chemosensitizers described to date may be grouped into six broad categories: (1) calcium channel blockers; (2) calmodulin antagonists; (3) noncytotoxic anthracycline and Vinca alkaloid analogs; (4) steroids and hormonal analogs; (5) miscellaneous hydrophobic cationic compounds; and (6) cyclosporines. Although these compounds share only broad structural similarities, most are extremely lipophilic, and those in the first five groups are all heterocyclic, amphipathic substances (Ford, J. M., et al., Pharmacol. Rev. 42:155 (1990)).